Detection of CCL25 and the correlation between CCL25, CCL28, IL-7, and TSLP in human breast milk

Highlights

• CCL25, a chemokine, was detected in human breast milk for the first time.

• CCL25 and TSLP levels were significantly higher in colostrum than in mature milk.

• CCL28 and IL-7 levels in colostrum showed a positive correlation.

• The level of parity has no effect on the CCL25, CCL28, TSLP and IL-7 levels.

Abstract

In this study, CCL25, a chemokine that contributes to the immunological function of the thymus and intestines, was detected in human breast milk (HBM) for the first time. We then focused on the correlations of CCL25 with CCL28, TSLP, and IL-7, which were predicted to interact with CCL25 in HBM. We also compared their levels between primiparous and multiparous women. A total of 53 parturient women were recruited. Their HBM was collected during 0–5 days and at 1 month after parturition and the CCL25, CCL28, IL-7, and TSLP levels in the HBM were analyzed using ELISA. The results showed that CCL25 and TSLP levels were significantly higher in colostrum than in mature milk. Moreover, CCL28 and IL-7 levels in colostrum showed a positive correlation. These results indicate that CCL28 and IL-7 in colostrum might interact positively with each other when produced in the mammary glands during lactation. The findings also suggest that the level of parity has no effect on their levels in HBM. In conclusion, our results clarify that CCL25 is present in HBM and that the concentrations of CCL25 and TSLP are higher in colostrum than in mature milk. Moreover, the production of CCL28 and IL-7 might be closely correlated in human colostrum.

Introduction

Human breast milk (HBM) contains a great variety of immunologically active substances, such as immunoglobulins including secretory immunoglobulin A (sIgA), growth factors, lactoferrin, and human milk oligosaccharides. These substances prevent bacterial infection and inflammation, and promote the development of immune organs and their immunological function, while also facilitating intestinal microbial colonization in neonates (Ballard, 2013, Gila-diaz et al., 2019, Nolan et al., 2019). Many different types of cytokines and chemokines are also present in breast milk (BM). However, little is known about their physiological and immunological functions for neonates. Gila-Diaz et al. (2019) suggested that HGF in BM is required for intestinal maturation in neonates. Other studies also demonstrated that IL-7 in BM is involved in thymus development in neonates (Ngom et al., 2004, Hossny et al., 2020). Against this background, it is considered that other cytokines and chemokines in BM might also play important roles in the development of neonatal immune organs and their functions.

Among several chemokines, CCL25 (C-C motif chemokine ligand 25) is expressed mainly in thymus, small intestine, and colon (Bekker et al., 2015), for which the only receptor is CCR9. CCR9 is expressed in early thymic progenitors and contributes to their migration into the thymus along with CCR7, the receptor of CCL19 and CCL21 (Zlotoff et al., 2010, Krueger et al., 2010). Furthermore, CCL25–CCR9 interactions in the thymus may be involved in the cortical localization of early thymocytes (Uehara et al., 2006, Lancaster et al., 2018). CCL25 also maintains the intestinal immune system by recruiting CCR9⁺ T cells (Kunkel et al., 2000, Gao et al., 2021) and CCR9⁺ IgA-antibody-producing cells (IgA-APCs) into the intestine (Bowman et al., 2002, Zhiming et al., 2018). Thus, CCL25 in BM may be crucial for the development of immune organs, along with HGF and IL-7. However, it has yet to be confirmed whether CCL25 is present in BM. Therefore, as the primary aim of this study, we attempted to detect CCL25 in HBM.

CCL28, IL-7, and TSLP are chemo/cytokines that are predicted to interact with CCL25. CCL28 (C-C motif chemokine ligand 28) is a chemokine exhibiting physiological functions similar to CCL25 and is mainly expressed in mucosal epithelial cells in salivary glands, colon, and lactating mammary glands. CCL28 attracts CCR10⁺ IgA-APCs and maintains the mucosal immune system (Matsuo et al., 2018, Zhiming et al., 2018). CCL28 in mammary tissue has also been reported to contribute to the transfer of IgA antibodies from the mother to the neonate by attracting CCR10⁺ IgA-APCs (Wilson and Butcher, 2004, Morteau et al., 2008). CCL28 also exhibits antibacterial activity against certain bacterial classes such as Candida albicans (Hieshima et al., 2003). CCL28 has already been detected in mouse and human milk (Hieshima et al., 2003, Boumahrou et al., 2012) and in BM it may contribute to mucosal immune functions in newborns. IL-7 and TSLP are cytokines that have been reported to be involved in the expression and production of CCL25. IL-7 and TSLP are expressed in the thymus and intestinal epithelial cells (Watanabe et al., 1995, Taylor et al., 2009). In addition, Ponte et al. (2017) revealed that the expression of CCL25 and CCL28 was promoted in small intestine treated with IL-7. A recent study also demonstrated that TSLP enhanced Treg cell differentiation induced by CCL25 (Manisha et al., 2020). IL-7 and TSLP have also been detected in HBM (Ngom et al., 2004, Hossny et al., 2020, MacFarlane et al., 2010). Therefore, interactions among CCL25, CCL28, IL-7, and TSLP in BM may affect their production in mammary glands, the development of immune organs, and their functions in neonates. Indeed, previous studies confirmed interactions among several BM cytokines (Böttcher et al., 2000, Oddy et al., 2003, Jarvinen et al., 2015). However, the interactions between CCL25, CCL28, IL-7, and TSLP in BM remain unknown. Thus, as a secondary aim of this study, we examined the correlations between CCL25, CCL28, IL-7, and TSLP levels in HBM. Additionally, to investigate whether parity affects the composition of BM, we compared CCL25, CCL28, IL-7, and TSLP levels between primiparous and multiparous women.